We wish to continue our studies of acute ACTH action in adrenal cortex cells to elucidate the sequence of molecular changes which cause an increase in the rate of formation of pregnenolone from cholesterol catalyzed by mitochondrial cytochrome P-450 in the initial and rate-limiting step in adrenal corticosteroidogenesis. We shall continue our characterization of a rapidly induced protein that we have discovered; the ACTH-dose response and the kinetics of appearance of this protein parallel very closely the increase in corticosteroid synthesis produced by ACTH. We shall try to describe its physical properties more closely and to determine its physiological function, i.e. the mechanism by which it causes an increase in pregnenolone formation. We shall use radioisotope labeling, e.g. incorporation of radiolabeled amino acids, acetate, 32PO4= etc., and gel electrophoresis to try to detect other cellular components newly synthesized or modified in response to ACTH. Subsequently we shall use chromatographic techniques (molecular sieving, ion exchange, affinity as well as high pressure liquid) to try to isolate such molecules. We shall also devote some of our attention to the proximal changes which are brought about in the mitochondria to effect this increase in rate, trying to detect changes in the lipid composition or distribution in mitochondrial membranes. We shall use epr spin-label spectroscopy to try to detect overall fluidity changes which might bring about different degrees of association of phospholipids, steroids, or auxillary electron transferring proteins with the cytochrome P-450scc. We hope to dissect out the sequence of events that allows this communication between the polypeptide and the steroid hormone systems.